Electronic Journal of Ichthyology

November, 2007 2: 46-60

THE EFFECT OF CADMIUM AND CADMIUM/COPPER MIXTURE DURING THE

EMBRYONIC DEVELOPMENT ON DEFORMATION OF COMMON CARP LAVAE

Katarzyna ugowska
Dept. of Animal Physiology University of Podlasie, Prusa 12, 08-110 Siedlce, Poland
e-mail: kongo@ap.siedlce.pl

Accepted: August, 2 2007

Abstract: The aim of present study was to estimate the effect of single and binary combina-
tions of cadmium with copper during the embryonic development on hatching success and
quality of common carp larvae. The results were compared to those obtained after copper ex-
posure, during the same experiment (Witeska and ugowska 2004).
The study was done on common carp newly hatched larvae, the embryonic development of
which took place at 0.2 mg/dm3 of cadmium (Cd group) and 0.2 (0.1+0.1) mg/dm3 mixture of
cadmium and copper (CdCu group), or in clean tap water (control K group). Newly hatched
larvae were counted and inspected, and for 20 days from hatching reared under control condi-
tions. The results show that cadmium (0.2 mg/dm3) reduced the success of hatching and
caused an increase of number of deformed larvae among the newly hatched ones. The results
obtained in the same experiment after copper exposure (Cu 0.2 mg/dm3) (Witeska and
ugowska 2004) indicate that toxic effect of this metal on hatching and quality of larvae was
stronger than the effect of cadmium. No differences in effect of cadmium exposure (0.2
mg/dm3) and its co-exposure with more toxic copper (0.1 mg/dm3 Cd+0.1 mg/dm3 Cu) on
hatchability and frequency of deformations were observed. These results indicate metal inter-
action, which in this case probably was antagonistic. Metal exposure decreased survival of fed
and starved normal and deformed larvae. Fish died gradually just after hatching. The com-
parison of effect of cadmium and Cd+Cu mixture shows that the mixture of both metals was
more toxic than cadmium alone, but not stronger than copper alone. These data suggest that
effect of metal mixture can be additive. Detailed classification of deformed larvae in the pre-
sent study showed that cadmium alone and in co-exposure with copper caused the same types
of larval deformations: vertebral curvatures, body and yolk sac deformations. Under optimal
conditions in laboratory culture, vertebral deformations may recover.

Keywords: cadmium, metal mixture, embryonic development, fish, deformations

Introduction Most studies of the effects of metals on fish

Cadmium is one of the metals most
commonly occurring in polluted natural
waters which may affect the early stages of
fish development. The data by various au-
thors show the adverse effect of cadmium
especially on hatchability (Williams and
Holdway 2000, Calta 2001, Gonzales-
Doncel et al. 2003, Sikorska and
ugowska 2005) and quality of larvae
(Weis and Weis 1977, Woodworth and
Pascoe 1982, Witeska et al. 1995, Hallare
et al. 2005, Fraysse et al. 2006).
concern exposures to single metal. Polluted
water bodies, however, usually contain ele-
vated levels of various metals. Many data
show that certain metals affect accumulation
of the others in fish. It seems that interactions
among various metals are related to their
competitive uptake from the environment, and
different distribution in fish tissues. Interac-
tions among metals may be different, and
therefore the effects of their various mixtures
on fish survival may also differ. The effect of
mixtures of two or more chemicals is com-
monly referred to either as additive, synergis-

46
ugowska K., 2007
tic or antagonistic (Jezierska and Witeska
2001).
In natural polluted waters cadmium is
very often accompanied by copper. There-
fore, it is interesting to evaluate combined
effect of both metals on survival and de-
velopment of larvae.
Toxicity of metal mixtures to fish dur-
ing development and growth was studied
by Kazlauskien et al. (1996),
Kazlauskien et al. (1999), Petrauskien
(1999), and Stokus et al.(1999), who tried
to simulate natural conditions of contami-
nated water bodies of Lithuania. However,
the data on the effects of metal mixtures on
embryonic development and hatched lar-
vae of fish are very scarce.
Kazlauskien and Stasinait (1999)
evaluated effects of heavy metal mixture
(Cu, Zn, Cr, Ni, Fe) on the eggs and
growth of rainbow trout larvae. Authors
revealed that even low concentrations of
the toxicant significantly decreased vitality
of eggs and larvae. Eaton (1973) exposed
Pimephales promelas to copper, cadmium
and zinc mixture and estimated growth,
spawning and hatching success. The mean
terminal weight of both males and females
was less in the highest metal mixture con-
centration. Spawning in all higher concen-
trations (above 0.01 mg/dm3 of each metal
in mixture) was greatly reduced (78% or
more) as compared to control results. Au-
thor concluded that it was possible to de-
termine whether or not the metals in the
mixture were additive in their effects, be-
cause each of them had one or more spe-
cific and identifiable toxic action.
The aim of presented study was to esti-
mate the effect of single and binary combi-
nations of cadmium with copper during the
embryonic development on hatching suc-
cess and quality of common carp larvae.
The results were compared to those ob-
tained after copper exposition, during the
same experiment conducted on the same
fish and presented by Witeska and
ugowska (2004).
Effect of Cadmium
Materials and Methods
The study was done on common carp
newly hatched larvae, the embryonic devel-
opment of which took place at 0.2 mg/dm3 of
cadmium (Cd group) and 0.2 (0.1+0.1)
mg/dm3 mixture of cadmium and copper
(CdCu group) or in clean tap water (control
K group). Metal solutions were made using
CdCl22 H2O and CuSO45H2O. The ex-
periments were done in 3 series.
Newly hatched larvae were counted and in-
spected. The percent of hatchability was cal-
culated as a number of hatched larvae per ini-
tial number of incubated eggs. Among freshly
hatched larvae the share of deformed ones was
evaluated. Each deformed larva was examined
and classified according to the types of mal-
formations using the catalog by Jezierska et al.
(2000).
For 20 days from hatching, the larvae were
reared under control conditions: dechlorinated
tap water, temperature 22C, oxygen satura-
tion about 80%, pH 7.8, hardness 230 mg/dm3
as CaCO3.
In all three series, the larvae were reared
separately in the 50 cm3 glass vessels of 7.5
cm diameter. Each vessel was individually
marked in order to identify each larva. During
the experiments, the fish were starved. Their
morphology was examined daily, and mortal-
ity was recorded.
The observations were made using a thick
slide with a concave chamber, which was
filled with clean water. Each larva was care-
fully placed in the chamber and observed us-
ing binocular (magnification 1.5x1.6) con-
nected with camera, and computer image
analysis system MultiScan. The observations
were recorded in photographs.
Additionally, in series III, the fed (twice a
day with Artemia sp. naupli), and starved lar-
vae were reared in the 100 ml glass vessels, at
the density of 20 fish per vessel. Normal and
deformed larvae from K group, and deformed
ones from Cd and CdCu groups were reared
separately. Survival was recorded daily.
Results
Metal exposure caused significant reduc-
tion of hatching rate and numerous body mal-
47
ugowska K., 2007 Effect of Cadmium

formations (Table 1). No differences in
both parameters were observed between
Cd and CdCu-exposed groups.
In both Cd and CdCu-exposed groups
the same four types of body malformations
were distinguished (Table 2). The changes
in yolk sac were the most frequent, ob-
served in 13 (Cd group) and 14 (CdCu
group) larvae, respectively. In other 9 and
3 individuals from these groups vertebral
deformations were observed, and in 5 and
4 fish body shortening.
Normal fed larvae from the control
group (Figure 1), reared under optimum
conditions showed very high survival rate,
while deformed ones (from control and Cd
or CdCu exposure) kept dying over entire
experimental period. Only 6 deformed in-
dividuals from the control, 3 from Cd-
exposed and 2 CdCu-exposed groups sur-
vived until the end of experiment. Most of
the starved control larvae survived 13 days
post hatching, but before the 20 day all
larvae quickly died (Figure 2). The de-
formed larvae from Cd exposure (single or
binary with copper) gradually died from
the beginning of the experiment. Until the
13 day in group Cd survived up to 5 (Cd
group) and 2 (CdCu group) larvae respec-
tively. All larvae from Cd exposure died
before 19 and CdCu before 17 day. De-
tailed observation of individuals provided
more information about changes in body mor-
phology of starved deformed carp larvae. In
the Cd groups (Figure 2a-c) all the larvae
showing G-type deformations died first, and
only 1 of them partly recovered. Similarly,
also E-type larvae died soon after hatching,
and only 2 fish in series I completely recov-
ered, and survived until 15 and 19 day. The B
deformation was reversible completely only in
1 series II fish, partly in 2 (series II, II) fish,
and B-type larvae survived until the 5 (series
I) and 13 (series II) day post hatch. On the
contrary, A deformation was reversible in all
larvae, and these fish showed the longest sur-
vival.
In CdCu exposed groups the larvae show-
ing G and E-type deformations died soon after
hatching. Among them, only 3 E deformed
fish (1 in each series) partly recovered, and all
larvae died until the 14 day from hatching.
The B deformation was reversible in only 2
fish (1 complete), while other fish died be-
tween 2 and 9 after hatching. In all series, the
A deformation was reversible, and larvae sur-
vived until 15 (series I) and 17 (series II, III)
day post hatching. Figure 4 shows some ex-
amples of gradual recovery of deformed lar-
vae observed in the present study. Fish in the
Figure 4a and 4c after hatching showed sco-
liosis.

Table 1. Hatching rate and quality of newly hatched larvae.

Series Group % of hatch % of deformed larvae
K 89.9 14.6
I Cd 73.3* 25.5*
CdCu 61.7* 24.2
K 71.7 14.2
II Cd 46.9* 33.3*
CdCu 46.7* 28.4
K 76.7 4.4
III Cd 58.3* 35.2*
CdCu 53.3* 30.7*
(* values significantly different from control, U-Mann-Whitney test, p 0.05, n=5)

48
ugowska K., 2007 Effect of Cadmium

49
ugowska K., 2007 Effect of Cadmium

50
ugowska K., 2007 Effect of Cadmium

Figure 1. Survival of the fed carp larvae.

H - hatching
Kn normal larvae from the control
Kd deformed larvae from the control
Cd d deformed larvae from cadmium exposure
CdCu d deformed larvae from cadmium and copper mixture

Figure 2. Survival of starved carp larvae.

H - hatching
Kn normal larvae from the control
Kd deformed larvae from the control
Cd d deformed larvae from cadmium exposure
CdCu d deformed larvae from cadmium and copper mixture
I series I, II - series II, III series III

51
ugowska K., 2007 Effect of Cadmium

Figure 3a. The changes in body morphology of starved deformed carp larvae.
(Cd, series I)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

Figure 3b. The changes in body morphology of starved deformed carp larvae.
(Cd, series II)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

52
ugowska K., 2007 Effect of Cadmium

Figure 3c. The changes in body morphology of starved deformed carp larvae.
(Cd, series III)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

Figure 3d. The changes in body morphology of starved deformed carp larvae.
(CdCu, series I)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

53
ugowska K., 2007 Effect of Cadmium

Figure 3e. The changes in body morphology of starved deformed carp larvae.
(CdCu, series II)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

Figure 3f. The changes in body morphology of starved deformed carp larvae.
(CdCu, series III)
H hatching
deformed larva
- gradual recovery
normal larva
+ - death

54
ugowska K., 2007 Effect of Cadmium

Figure 4a. Complete gradual recovery of the starved larvae exposed to Cd during em-
bryonic development.

55
ugowska K., 2007
Figure 4b. Partial gradual recovery of the starved larvae exposed to CdCu during em-
bryonic development.
Curvature started to decrease already on
the first day post hatching, and disappeared
until 6 and 5 day, respectively, when the
larvae looked quite normally. But in some
cases the recovery wasnt complete. Newly
hatched larvae from CdCu (Figure 4b)
showed C-shaped body, and their spine
started to straighten gradually, beginning
from the first day post hatching. On the 3
day fish still sowed slightly curved tail
region which remained curved until the
death of the larvae.
Discussion
The results show that cadmium (0.2
mg/dm3) reduced the success of hatching
(Table 1). Decrease of hatching rate with
increase of cadmium concentration within
the range of 0.001-0.02 mg/dm3 Cd was
reported by Witeska et al. (1995), and in
0.2-0.7 mg/dm3 Cd by Sikorska and
ugowska (2005). Calta (2001) observed
Effect of Cadmium
that at 0.01 mg/dm3 of Cd only 46% of mirror
carp larvae hatched. Cadmium in concentra-
tions 3.3 and 33.3 mg/dm3 reduced number of
hatched Melanotaenia fluviatilis larvae (Wil-
liams and Holdway 2000). Gonzales-Doncel
et al. (2003) observed a decrease of Danio
rerio hatching rate at Cd concentrations of 20,
40, and 80 mg/dm3, and Hallare et al. (2005)
at 0.25-10 mg/dm3.
Exposure to Cd caused also an increase in
the number of deformed larvae among the
newly hatched ones. The results of the present
study confirm data obtained by other authors.
Witeska et al. (1995) reported that 47% of
larvae were deformed after exposure to 0.02
mg mg/dm3 of Cd in comparison to only 1%
in control. The proportion of mirror carp de-
formed larvae increased with increasing of
cadmium concentration from 0.002 to 0.05
mg/dm3 of Cd was observed by Calta (2001).
56
ugowska K., 2007
Fig. 4c. Complete gradual recovery of the starved larvae exposed to CdCu during embryonic
development.
The results obtained in the same ex-
periment after exposure to 0.2 mg/dm3 of
Cu (Witeska and ugowska 2004) indicate
that toxic effect of this metal on hatching
and quality of larvae was stronger than 0.2
mg/dm3 of Cd: the hatching rates in Cu
groups were 60, 38.3 and 40%, while in the
present study in Cd groups: 73.3, 46.9 and
58.3%, respectively. Percentage of de-
formed larvae after Cu exposure were: 41,
64 and 66%, while in Cd exposure: 25.5,
33.3 and 30.8%. Also 48 hours LC50 of
heavy metals for embryos of Paralichthys
Effect of Cadmium
olivaceus exposed to Cu, Cd, Cr and Pb indi-
cated that copper was the most toxic to em-
bryos (Wu et al. 1990). This indicates that
copper is more toxic than cadmium at the
same concentration.
Different toxicity of cadmium and copper
on embryonic development can be a result of
different uptake and accumulation of metals
by the eggs. According to Beatie and Pascoe
(1978), Michibata (1981), Burinson et al.
(2006), the most of cadmium is accumulated
in the egg outer shell chorion, and only
small amount in the embryo. However, no
57
ugowska K., 2007
data are available on copper accumulation,
which as an essential metal, and is proba-
bly taken up into the embryo.
No differences in effect of cadmium ex-
posure (0.2 mg/dm3) and co-exposure with
more toxic copper (0.1 mg/dm3 Cd+0.1
mg/dm3 Cu) on hatchability and frequency
of deformations were observed. These re-
sults indicate metal interaction, which in
this case is probably antagonistic, as it was
suggested by Jezierska and Witeska
(2001). Also the results obtained by
ugowska (2005) showed that the effects
of cadmium and copper mixture on various
parameters of embryonic development
(such as swelling, mortality of embryos,
deformations of larvae and yolk sac vol-
ume) were weaker than the effect of each
metal separately.
Detailed classification of deformed lar-
vae in the present study showed that cad-
mium alone and together with copper
caused the same types of larval deforma-
tions: vertebral curvatures, body and yolk
sac deformations. Exactly the same types
of deformations of carp larvae were ob-
served by Witeska and ugowska (2004)
after 0.2 mg/dm3 copper exposure.
These types of malformations are not
cadmium- (or any other metal) specific,
and they were observed also in fish ex-
posed to other heavy metals. Similar mal-
formations such as: deformed yolk sac,
curved spinal column, enlarged head and
edema were described by Jezierska and
Somiska (1997) in copper-exposed
common carp larvae. Jezierska and
Grzyska (1998) observed shortening and
curvature of caudal part, vertebral malfor-
mations, and yolk sac malformations in
common and grass carp after copper (0.2
and 0.3 mg/dm3) and lead (5.0 mg/dm3)
exposures. C-shape body after mercury
exposure in Oryzias latipes were described
by Heisinger and Green (1975). Klein-
MacPhee et al. (1984) observed shortened,
twisted bodies and yolk sac malformations
in winter flounder exposed to silver. Lor-
dosis, twisted tails and inclusions in yolk
Effect of Cadmium
sac in rainbow trout were reported by Stasin-
ait (1999) after Ni, Zn, Pb, Cd, Fe and Mn
exposure.
In laboratory culture, the hatched lar-
vae were reared in clean tap water. Therefore,
observed survival of larvae was affected only
by the conditions of the embryonic develop-
ment, when eggs were exposed to metals.
Metal exposure reduced survival of fed
(Figure 1) and starved (Figure 2) normal and
deformed larvae. Fish died gradually after
hatching. Similar, but slightly stronger toxic
effect of copper on survival of larvae was ob-
served by Witeska and ugowska (2004).
The comparison of effect of cadmium and
mixture cadmium and copper shows that the
mixture of both metals was more toxic than
cadmium alone, but not stronger than copper
alone. According to various data presented by
Jezierska and Witeska (2001), these results
suggest that effect of metal mixture can be
additive.
Similar interaction between Cd and Cu in
mixture was observed by Jezierska et al.
(2006) on feeding activity of common carp
larvae. Exposure to copper in mixture with
cadmium impaired food uptake much more
than in cadmium alone.
Detailed observations and assessment of
deformed larvae in laboratory culture (Fig-
ure3) showed that complex malformations
caused death soon after hatching, and only
spine curvatures were reversible. Similar ef-
fect after 0.2 mg/dm3 Cu exposure was ob-
served by Witeska and ugowska (2004), and
recovery was possible only in larvae with ver-
tebral malformations. Thus, under optimal
conditions some deformations of larvae, par-
ticularly curvatures of the spinal column, may
recover.
References
Beattie, J.H. Pascoe D. (1978). Cadmium
uptake by rainbow trout, Salmo gairdneri
eggs and alevins. J. Fish. Biol., 13: 631-
637.
Burinson, B.K., Meintel, T., Playle, R., Piet-
rock, M., Wienke, A., Steinberg, Ch.E.W.
58
ugowska K., 2007
(2006). Cadmium accumulation in ze-
brafish (Danio rerio) eggs is modu-
lated by dissolved organic matter
(DOM). Aquat. Toxicol., 79: 185-191.
Calta, M. (2001). Effect of aqueous cad-
mium on embryos and larvae of mirror
carp. Indian J. of Animal Sci., 71 (9):
885-888.
Eaton J.G., (1973). Chronic toxicity of
copper, cadmium and zinc mixture to
the fathead minnow Pimephales pro-
melas (Rafinesque). Wat. Res. 7: 1723-
1736.
Fraysse, B., Mons, R., Garric, J. (2006).
Development of zebrafish 4-day em-
bryo-larval bioassay to assess toxicity
of chemicals. Ecotox. and Environ.
Saf., 63: 253-267.
Gonzles-Doncel, M., Larrea, M.,
Snchez-Fortn, S., Hinton, D.E.
(2003). Influence of water hardening of
the chorion on cadmium accumulation
in medaka (Oryzias latipes) eggs.
Chemosphere, 52: 75-83.
Hallare, A.V., Schirling, M.,
Luckenbach, T., Khler, H.-R.,
Triebskorn, R. (2005). Combined ef-
fects of temperature and cadmium on
developmental parameters and bio-
markers responses in zebrafish (Danio
rerio) embryos. J. Thermal Biol., 30: 7-
17.
Heisinger, J.F., Green, W. (1975). Mer-
curic chloride uptake by eggs of the
ricefish and resulting teratogenic ef-
fects. Bull. Environ. Contam. Toxicol.,
14: 665-673.
Jezierska, B., Grzyska, K. (1998).
Malformations of cyprinid embryos
and larvae after exposure to copper and
lead. 2nd International Conference Tace
elements Effects on Organisms ahd
Environment, Cieszyn Poland, 23-26
June.
Jezierska, B., ugowska, K., Sarnowski,
P., Witeska, M. (2006). The effect of
short-term water contamination with
heavy metals on ford uptake rate of
59
Effect of Cadmium
common carp larvae. Electornic Journal of
Ichthyology, 1: 1-11.
Jezierska, B., ugowska, K., Witeska, M.,
Sarnowski, P. (2000). Malformations of
newly hatched common carp larvae. Elec-
tronic Journal of Polish Agricultural Uni-
versities, Fisheries, 3 (2).
Jezierska, B., Somiska, I. (1997). The ef-
fect of copper on common carp (Cyprinus
carpio L.) during embryonic and postem-
bryonic development. Pol. Arch. Hydro-
biol., 44: 261-272.
Jezierska, B., Witeska, M. (2001). Metal
toxicity to fish. Wydawnictwo AP.
Siedlce. 318 pp.
Kazlauskien, N., Sveceviius, G., Vosy-
lien, M.Z. (1999). The use of rainbow
trout (Oncorhynchus mykiss) as a test-
object for evaluation of the water quality
polluted with heavy metals. Heavy Metals
in the Environment: An Integrated Ap-
proach, (Lovejoy, D.A., Ed.) Vilnius, 231-
233.
Kazlauskien, N., Stasinait, P. (1999).
The lethal and sublethal effect of heavy
metal mixture on rainbow trout (On-
corhynchus mykiss) in its early stages of
development. Acta. Zool. Lithuanica. Hy-
drobiol., 1: 47-54.
Kazlauskien, N., Burba, A., Sveceviius,
G. (1996). Reactions of hydrobionts on ef-
fect of mixture of five galavanic heavy
metals. Ekologija, 4: 56-59.
Klein-MacPhee, G., Cardin, J.A., Berry,
W.J. (1984). Effects of silver on eggs and
larvae of the winter flounder. Trans. Am.
Fish Soc., 113: 247-251.
ugowska, K. (2005). Wpyw miedzi i
kadmu na przebieg rozwoju
embrionalnego i jako wyklutych larw
karpia (Cyprinus carpio L.) [The effect of
copper and cadmium on embryonic devel-
opment and quality of hatched carp larvae
(Cyprinus carpio L.)]. Ph.D. Thesis.
Academy of Podlasie, Siedlce. 127pp[In
Polish]
ugowska K., 2007 Effect of Cadmium

Michibata, H. (1981). Uptake and distri- killifish, Fundulus heteroclitus. J. Fish

bution of cadmium in the egg of the
teleost, Oryzias latipes. J. Fish Biol.,
19: 691-696.
Petrauskien, L. (1999). Effects of novel
environment on rainbow trout exposed
to copper. Acta Zool. Lituanica. Hy-
drobiol., 2: 95-102.
Sikorska, J., ugowska, K. (2005).
Wpyw kadmu na rozwj embrionalny
karpia (Cyprinus carpio L.) [The effect
of cadmium on embryonic develop-
ment of carp (Cyprinus carpio L.)].
Kom. Ryb., 3: 6-8 [In Polish].
Stasinait, P. (1999). Long-term heavy
metal mixture toxicity to embryos and
alevins of rainbow trout (Oncorhyn-
chus mykiss). Acta Zool. Lituanica.
Hydrobiol., 2: 40-45.
Stokus, L., Stokus, A., Kazlauskien,
N. (1999). The use of early life stages
of zebrafish (Brachydanio rerio) for
toxicity evaluation of five heavy metals
(Cu, Ni, Fe, Zn, Cr) in water solutions.
In: Heavy Metals in the Environment:
An integrated approach, (Lovejoy,
D.A., Ed.) Vilnius, 234-238.
Weis, J.S., Weis, P. (1977). Effects of
heavy metals on development of the
Biol., 11: 49-54.
Williams, N.D., Holdway, D.A. (2000). The
effects of pulse-exposed cadmium and
zinc on embryo hatchability, larval devel-
opment, and survival of Australian crim-
son spotted rainbow fish (Melanotaenia
fluviatilis). Environ. Toxicol., 15 (3): 165-
173.
Witeska, M., Jezierska, B., Chaber, J.
(1995). The influence of cadmium on
common carp embryos and larvae. Aqua-
culture, 129: 129-132.
Witeska, M., ugowska, K. (2004). The ef-
fect of copper exposure during embryonic
development on deformations of newly
hatched common carp larvae, and further
consequences. Electronic Journal of Polish
Agricultural Universities, Fisheries, 7 (2)
Woodworth, J., Pascoe, D. (1982). Cad-
mium toxicity to rainbow trout, Salmo
gairdneri Richardson: a study of eggs and
alevins. J. Fish Biol., 21: 47-57.
Wu, Y.L., Zhao, H.R., Hou, L.Y. (1990).
Effects of heavy metals on embryos and
larvae of flat fish Paralichthys olivaceus.
Ocean. Et Limnol. Sinica (Hai Yang Yu
Chao) HYHCAG, 21: 386-393.

===================================================================
Copies of the PDF file of this work have been deposited in the following publicly accessible libraries: 1.
National Museum of Natural History, Smithsonian Institution, Washington D.C. USA; 2. Natural History
Museum, London, UK; 3. California Academy of Sciences, San Francisco, California, USA; 4. Department of
Ichthyology, Museum National d'Histoire Naturelle, 75005 Paris, France; 5. Senckenberg Museum,
Frankfurt/Main, Germany; 6. National Museum of Natural History, Leiden, The Netherlands. 7. The Gitter-
Smolartz Library of Life Sciences and Medicine, Tel Aviv University, Israel; 8. The National and university
Library, Jerusalem, Israel; 9. Library of Congress, Washington, D.C. USA; 10. South African Institute for
Aquatic Biodiversity, Grahamstown, South Africa; 11. The National Science Museum, Tokyo, Japan; 12. The
Swedish Museum of Natural History, Stockholm, Sweden.

60